Recovery method for nucleic acid-containing solution used for sequencing on machine

Abstract

The present invention belongs to the field of gene sequencing, and particularly relates to a recovery method for a nucleic acid-containing solution used for sequencing on a machine. The method can fully utilize the feature of a high density of a nucleic acid-containing solution used for sequencing on a machine, and the density of the nucleic acid-containing solution will not obviously reduced after one time of use, and therefore a recovered nucleic acid-containing solution still has the capability of being loaded onto flow cells for two times, even three times for sequencing, thus improving the utilization rate of nucleic acid-containing solutions, realizing the purpose of recovery of nucleic acid-containing solutions, and avoiding the labor and material consumption caused by repeatedly preparing nucleic acids (libraries). When being used for sequencing, and if there is a requirement of repeated assays, the present method can directly perform sequencing again without the processes of preparing libraries and nucleic acid-containing solutions, thereby shortening the sequencing process and reducing the preparation time and the sequencing cost of samples. In addition, the present method can avoid insufficient data volumes in sequencing due to excessively small amounts of rare samples, thereby improving the utilization rate of rare samples and further improving the depth of sequencing.

Description

A method for recycling nucleic acid-containing solution for sequencing Technical Field

[0001] The present invention belongs to the field of gene sequencing, and in particular relates to a method for recycling a nucleic acid-containing solution used for on-machine sequencing. Background Art

[0002] With the rapid development and increasing recognition of biosequencing technology, high-throughput gene sequencing has been widely adopted in various fields, including scientific research, precision medicine, public health, and public safety. Driven by continuous innovation and R&D, the cost of massively parallel sequencing (MPS) has continued to decline. In the first half of 2022, Ultima Genomics released the UG100 sequencer, claiming to be capable of SE300 sequencing, reducing the cost of each WGS (whole genome sequencing, 100Gb of data) to $100. On September 30, 2022, Illumina released its latest NovaSeq X-Series sequencer, capable of PE150 sequencing, with a sequencing cost of $200 per WGS. In 2023, MGI released the DNBSEQ-T20x2 ultra-high-throughput sequencer, marking the entry of mainstream MPS sequencing platforms into the $100 per WGS era. As sequencing costs fall to affordable levels for the general public, this will significantly boost the application of high-throughput sequencing in areas such as infectious diseases, cancer prevention and treatment, and neonatal disease screening.

[0003] MGI, a leading domestic high-throughput sequencer company, utilizes its unique DNBSEQ sequencing technology across its various sequencing platforms. This technology involves three key elements: DNA nanoballs (DNBs), patterned arrays, and combined probe anchor synthesis (cPAS). DNA nanoballs (DNBs) are loose, "fuzzy," collections of 300-500 copies of single-stranded DNA using rolling circle amplification (RCA) technology, using circular single-stranded DNA as a template. They are the essential building blocks of DNBSEQ sequencing technology.

[0004] In DNBSEQ sequencing technology, DNA nanoballs must be prepared before each sequencing run. As shown in Figure 1, the DNA nanoballs are prepared using rolling circle amplification (RCA), an isothermal amplification reaction catalyzed by a DNA polymerase with strand displacement activity. Primers are paired with circular DNA templates from different biological samples. The DNA polymerase then extends the primers along the ring, continuously displacing previously generated extended strands, ultimately producing repetitive long single-stranded DNA products. This process uses only one strand of the double-stranded DNA as a template, preventing the accumulation of amplification errors and ensuring accuracy in subsequent sequencing.

[0005] After the DNB is prepared, it needs to be loaded into the slide to achieve stable cycle sequencing. As shown in Figure 2, the DNB mixture will first be passed into the surface of the sequencing slide, where a single DNB will bind to each sequencing site. During this process, the DNB settles on the slide surface due to gravity, and then attracts the positively charged sequencing sites under the action of Coulomb force, and then binds to the amino-modified sites on the surface under the action of van der Waals force. Finally, the DNB is protein-embedded under the action of a series of postload reagents to ensure that the DNB can be stably adsorbed on the sequencing site and will not loosen and fall due to subsequent frequent fluid flushing. Different types of sequencing reagents are then introduced to carry out the subsequent complete sequencing process.

[0006] During conventional DNB loading, once the DNBs have finished adsorbing to the slide, they are pushed out by the next pumped reagent and discarded into the waste area. Typically, the DNB mixture is used only once. If sequencing is repeated multiple times, DNBs must be prepared again. However, the used DNB mixture still contains DNBs equivalent to 90% of the initial loading concentration (calculated according to the DNBSEQ sequencing principle) and can be reloaded onto the slide, allowing it to be recycled and reused, improving sequencing efficiency.

[0007] Preparation of the DNB mixture often places high demands on the DNA template library, including library input and quality. For rarer samples such as forensic, archaeological, rare plant and animal, and cell-free DNA, or for sequencing types requiring complex preparation processes, such as single-cell sequencing, a fixed library size can only produce a fixed volume of DNBs. The amount of data generated by subsequent sequencing depends on the quality of the sequencing chip, the performance of the sequencing platform, and the operator's skill. If sample quality is poor or the sample size is insufficient, the prepared DNBs may not be sufficient to meet the required sequencing data volume. In this case, it is often necessary to recollect the sequencing sample and prepare the library, prepare DNBs again, and repeat the sequencing multiple times, or even change the sequencing platform until the usage requirements are met.

[0008] Furthermore, in some cases where engineers only need to perform repetitive testing and have low sequencing quality requirements, the quality of the DNA nanospheres is not particularly demanding. However, each commissioning still requires re-preparing the DNB mixture and even re-building the library, which increases the number of steps, materials, personnel, and time costs. Therefore, it is necessary to develop a method for recycling the nucleic acid-containing solution used for sequencing.

[0009] Summary of the Invention

[0010] The purpose of the first aspect of the present invention is to provide a method for recycling a nucleic acid-containing solution used for sequencing.

[0011] The second aspect of the present invention aims to provide a system for recycling nucleic acid-containing solutions for sequencing.

[0012] The third aspect of the present invention aims to provide a sequencing method.

[0013] The fourth aspect of the present invention aims to provide a sequencing system.

[0014] The fifth aspect of the present invention aims to provide a method for screening nucleic acids in a nucleic acid-containing solution having specific physical characteristics.

[0015] The sixth aspect of the present invention aims to provide a simulated solution containing nucleic acid solution.

[0016] In order to achieve the above object, the technical solution adopted by the present invention is:

[0017] A first aspect of the present invention provides a method for recycling a nucleic acid-containing solution for sequencing, comprising the following steps:

[0018] a1) pushing out the nucleic acid-containing solution in the sequencing tank of the sequencing slide to obtain a recovery solution containing the nucleic acid solution;

[0019] a2) Pushing the recovery solution containing the nucleic acid solution obtained in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing the nucleic acid solution.

[0020] Preferably, step a2) is repeated 0-3 times.

[0021] Preferably, the nucleic acid-containing solution in step a1) comprises any one of: DNB, circular DNA, fragmented DNA, and a sequencing complex containing a DNB template, a polymerase, and a sequencing primer.

[0022] Further preferably, the nucleic acid-containing solution in step a1) comprises: DNB.

[0023] Preferably, the nucleic acid-containing solution in step a1) further comprises: a loading buffer.

[0024] Preferably, the amount of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times, or 30 times or more the nucleic acid adsorption capacity of the sequencing tank of the sequencing slide (i.e., the number of nucleic acid adsorption sites in the sequencing tank).

[0025] Preferably, the pushing method in steps a1), and / or a2) comprises the step of pushing using a simulated liquid containing a nucleic acid solution.

[0026] Preferably, the pushing method in steps a1), and / or a2) comprises the following steps: pushing a simulation liquid containing a nucleic acid solution into the liquid inlet of the sequencing tank.

[0027] Preferably, the simulated liquid containing the nucleic acid solution is pushed in by a pipetting device.

[0028] Preferably, before the DNB simulation liquid is pushed into the liquid inlet of the sequencing tank, the method further comprises the following step: pushing gas into the liquid inlet of the sequencing tank, wherein the gas does not affect the performance of the DNB.

[0029] Preferably, the gas is pushed in by a pipetting device.

[0030] Preferably, the simulated liquid containing nucleic acid solution comprises an organic solvent, polyethylene glycol, and a buffer.

[0031] Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile.

[0032] Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000.

[0033] Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer.

[0034] Preferably, the pH of the buffer solution is 4.2-5.0.

[0035] Preferably, the concentration of the buffer solution is 0.1-0.4M.

[0036] Preferably, the final concentration of the buffer in the simulated solution containing nucleic acid solution is 62-88% (v / v).

[0037] Preferably, the final concentration of the polyethylene glycol in the simulated solution containing nucleic acid solution is 1-4% (m / v).

[0038] Preferably, the final concentration of the organic solvent in the simulated liquid containing nucleic acid solution is 10-30% (v / v).

[0039] Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides.

[0040] Preferably, in steps a1), and / or a2), the pushed nucleic acid-containing solution or the recovered liquid of the nucleic acid-containing solution is recovered by a pipetting device.

[0041] Preferably, the pipetting devices are each independently selected from at least one of a pipette, a syringe, and a syringe pump.

[0042] Preferably, the rolling circle amplification time of the DNB is 20-120 min.

[0043] A second aspect of the present invention provides a system for recycling a nucleic acid-containing solution for on-machine sequencing. The system is configured to perform a method for recycling a nucleic acid-containing solution for on-machine sequencing, the method comprising the following steps: a1) pushing the nucleic acid-containing solution out of a sequencing tank of a sequencing slide to obtain a recovered solution containing the nucleic acid solution;

[0044] a2) Pushing the recovery solution containing the nucleic acid solution obtained in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing the nucleic acid solution.

[0045] Preferably, step a2) is repeated 0-3 times.

[0046] Preferably, the nucleic acid-containing solution in step a1) comprises any one of: DNB, circular DNA, fragmented DNA, and a sequencing complex containing a DNB template, a polymerase, and a sequencing primer.

[0047] Further preferably, the nucleic acid-containing solution in step a1) comprises: DNB.

[0048] Preferably, the nucleic acid-containing solution in step a1) further comprises: a loading buffer.

[0049] Preferably, the amount of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times or 30 times greater than the nucleic acid adsorption capacity of the sequencing tank of the sequencing slide (i.e., the number of nucleic acid adsorption sites in the sequencing tank).

[0050] Preferably, the pushing method in steps a1), and / or a2) comprises the step of pushing using a simulated liquid containing a nucleic acid solution.

[0051] Preferably, the pushing method in steps a1), and / or a2) comprises the following steps: pushing a simulation liquid containing a nucleic acid solution into the liquid inlet of the sequencing tank.

[0052] Preferably, the simulated liquid containing the nucleic acid solution is pushed in by a pipetting device.

[0053] Preferably, before the simulation liquid containing the nucleic acid solution is pushed into the liquid inlet of the sequencing tank, the following step is further included: pushing gas into the liquid inlet of the sequencing tank, wherein the gas does not affect the performance of the DNB.

[0054] Preferably, the gas is pushed in by a pipetting device.

[0055] Preferably, the simulated liquid containing nucleic acid solution comprises an organic solvent, polyethylene glycol, and a buffer.

[0056] Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile.

[0057] Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000.

[0058] Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer.

[0059] Preferably, the pH of the buffer solution is 4.2-5.0.

[0060] Preferably, the concentration of the buffer solution is 0.1-0.4M.

[0061] Preferably, the final concentration of the buffer in the simulated solution containing nucleic acid solution is 62-88% (v / v).

[0062] Preferably, the final concentration of the polyethylene glycol in the simulated solution containing nucleic acid solution is 1-4% (m / v).

[0063] Preferably, the final concentration of the organic solvent in the simulated liquid containing nucleic acid solution is 10-30% (v / v).

[0064] Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides.

[0065] Preferably, in steps a1), and / or a2), the pushed nucleic acid-containing solution or the recovered liquid of the nucleic acid-containing solution is recovered by a pipetting device.

[0066] Preferably, the pipetting devices are each independently selected from at least one of a pipette, a syringe, and a syringe pump.

[0067] Preferably, the rolling circle amplification time of the DNB is 20-120 min.

[0068] Preferably, the system comprises:

[0069] a pushing module, which is used for pushing the nucleic acid-containing solution, the simulated solution containing the nucleic acid solution, the recovered solution containing the nucleic acid solution, and / or the gas; and

[0070] The recovery module is used for recovering the pushed nucleic acid-containing solution and / or the recovery liquid of the nucleic acid-containing solution.

[0071] Preferably, the pushing module comprises a pipetting device.

[0072] Preferably, the pushing module further comprises a storage container.

[0073] Preferably, the storage container is used for storing a nucleic acid-containing solution, a simulated liquid containing a nucleic acid solution, and / or a gas.

[0074] Preferably, the recovery module comprises a pipetting device.

[0075] Preferably, the recovery module further comprises a recovery container (such as a separate recovery cabin).

[0076] Preferably, the recovery container is used to store a recovery liquid containing a nucleic acid solution.

[0077] Preferably, the system is an automated system.

[0078] Preferably, the device in the pushing module and / or the recovery module is an automated device.

[0079] The third aspect of the present invention provides a sequencing method comprising the following steps:

[0080] a1) pushing out the nucleic acid-containing solution in the sequencing tank of the sequencing slide to obtain a recovery solution containing the nucleic acid solution;

[0081] a2) pushing the recovered solution containing the nucleic acid solution obtained in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovered solution containing the nucleic acid solution;

[0082] 3) Sequencing the nucleic acids in the nucleic acid-containing solution in the sequencing tank of the sequencing slide described in a1) and / or a2).

[0083] Preferably, step a2) is repeated 0-3 times.

[0084] Preferably, the nucleic acid-containing solution in step a1) comprises any one of: DNB, circular DNA, fragmented DNA, and a sequencing complex containing a DNB template, a polymerase, and a sequencing primer.

[0085] Further preferably, the nucleic acid-containing solution in step a1) comprises: DNB.

[0086] Preferably, the nucleic acid-containing solution in step a1) further comprises: a loading buffer.

[0087] Preferably, the amount of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times or 30 times greater than the nucleic acid adsorption capacity of the sequencing tank of the sequencing slide (i.e., the number of nucleic acid adsorption sites in the sequencing tank).

[0088] Preferably, the pushing method in steps a1), and / or a2) comprises the step of pushing using a simulated liquid containing a nucleic acid solution.

[0089] Preferably, the pushing method in steps a1), and / or a2) comprises the following steps: pushing a simulation liquid containing a nucleic acid solution into the liquid inlet of the sequencing tank.

[0090] Preferably, the simulated liquid containing the nucleic acid solution is pushed in by a pipetting device.

[0091] Preferably, before the simulation liquid containing nucleic acid solution is pushed into the liquid inlet of the sequencing tank, the following step is further included: pushing gas into the liquid inlet of the sequencing tank, wherein the gas does not affect the performance of the nucleic acid in the nucleic acid solution.

[0092] Preferably, the gas is pushed in by a pipetting device.

[0093] Preferably, the simulated liquid containing nucleic acid solution comprises an organic solvent, polyethylene glycol, and a buffer.

[0094] Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile.

[0095] Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000.

[0096] Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer.

[0097] Preferably, the pH of the buffer solution is 4.2-5.0.

[0098] Preferably, the concentration of the buffer solution is 0.1-0.4M.

[0099] Preferably, the final concentration of the buffer in the simulated solution containing nucleic acid solution is 62-88% (v / v).

[0100] Preferably, the final concentration of the polyethylene glycol in the simulated solution containing nucleic acid solution is 1-4% (m / v).

[0101] Preferably, the final concentration of the organic solvent in the simulated liquid containing nucleic acid solution is 10-30% (v / v).

[0102] Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides.

[0103] Preferably, in steps a1), and / or a2), the pushed nucleic acid-containing solution or the recovered liquid of the nucleic acid-containing solution is recovered by a pipetting device.

[0104] Preferably, the pipetting devices are each independently selected from at least one of a pipette, a syringe, and a syringe pump.

[0105] Preferably, the rolling circle amplification time of the DNB is 20-120 min.

[0106] A fourth aspect of the present invention provides a sequencing system, wherein the system is configured to perform a sequencing method, the sequencing method comprising the following steps:

[0107] a1) pushing out the nucleic acid-containing solution in the sequencing tank of the sequencing slide to obtain a recovery solution containing the nucleic acid solution;

[0108] a2) pushing the recovered solution containing the nucleic acid solution obtained in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovered solution containing the nucleic acid solution;

[0109] 3) Sequencing the nucleic acids in the nucleic acid-containing solution in the sequencing tank of the sequencing slide described in a1) and / or a2).

[0110] Preferably, step a2) is repeated 0-3 times.

[0111] Preferably, the nucleic acid-containing solution in step a1) comprises any one of: DNB, circular DNA, fragmented DNA, and a sequencing complex containing a DNB template, a polymerase, and a sequencing primer.

[0112] Further preferably, the nucleic acid-containing solution in step a1) comprises: DNB.

[0113] Preferably, the nucleic acid-containing solution in step a1) further comprises: a loading buffer.

[0114] Preferably, the amount of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times or 30 times greater than the nucleic acid adsorption capacity of the sequencing tank of the sequencing slide (i.e., the number of nucleic acid adsorption sites in the sequencing tank).

[0115] Preferably, the pushing method in steps a1), and / or a2) comprises the step of pushing using a simulated liquid containing a nucleic acid solution.

[0116] Preferably, the pushing method in steps a1), and / or a2) comprises the following steps: pushing a simulation liquid containing a nucleic acid solution into the liquid inlet of the sequencing tank.

[0117] Preferably, the simulated liquid containing the nucleic acid solution is pushed in by a pipetting device.

[0118] Preferably, before the simulation liquid containing nucleic acid solution is pushed into the liquid inlet of the sequencing tank, the following step is further included: pushing gas into the liquid inlet of the sequencing tank, wherein the gas does not affect the performance of the nucleic acid in the nucleic acid solution.

[0119] Preferably, the gas is pushed in by a pipetting device.

[0120] Preferably, the simulated liquid containing nucleic acid solution comprises an organic solvent, polyethylene glycol, and a buffer.

[0121] Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile.

[0122] Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000.

[0123] Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer.

[0124] Preferably, the pH of the buffer solution is 4.2-5.0.

[0125] Preferably, the concentration of the buffer solution is 0.1-0.4M.

[0126] Preferably, the final concentration of the buffer in the simulated solution containing nucleic acid solution is 62-88% (v / v).

[0127] Preferably, the final concentration of the polyethylene glycol in the simulated solution containing nucleic acid solution is 1-4% (m / v).

[0128] Preferably, the final concentration of the organic solvent in the simulated liquid containing nucleic acid solution is 10-30% (v / v).

[0129] Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides.

[0130] Preferably, in steps a1), and / or a2), the pushed nucleic acid-containing solution or the recovered liquid of the nucleic acid-containing solution is recovered by a pipetting device.

[0131] Preferably, the pipetting devices are each independently selected from at least one of a pipette, a syringe, and a syringe pump.

[0132] Preferably, the rolling circle amplification time of the DNB is 20-120 min.

[0133] Preferably, the system comprises:

[0134] a pushing module, which is used for pushing the nucleic acid-containing solution, the simulated solution containing the nucleic acid solution, the recovered solution containing the nucleic acid solution, and / or the gas; and

[0135] a recovery module, which is used to recover the pushed nucleic acid-containing solution and / or the recovery liquid of the nucleic acid-containing solution; and

[0136] The sequencing module is used to sequence the nucleic acid in the nucleic acid-containing solution in the sequencing tank of the sequencing carrier.

[0137] Preferably, the pushing module comprises a pipetting device.

[0138] Preferably, the pushing module further comprises a storage container.

[0139] Preferably, the storage container is used for storing a nucleic acid-containing solution, a simulated liquid containing a nucleic acid solution, and / or a gas.

[0140] Preferably, the recovery module comprises a pipetting device.

[0141] Preferably, the recovery module further comprises a recovery container (such as a separate recovery cabin).

[0142] Preferably, the recovery container is used to store a recovery liquid containing a nucleic acid solution.

[0143] Preferably, the sequencing module comprises a sequencing device.

[0144] Preferably, the system is an automated system.

[0145] Preferably, the devices in the pushing module, the recovery module, and / or the sequencing module are automated devices.

[0146] A fifth aspect of the present invention provides a method for screening a nucleic acid in a nucleic acid-containing solution having specific physical characteristics, comprising the following steps:

[0147] a1) pushing out the nucleic acid-containing solution in the sequencing tank of the sequencing slide to obtain a recovery solution containing the nucleic acid solution, wherein the sequencing tank of the sequencing slide is capable of adsorbing nucleic acids in the nucleic acid-containing solution having the specific physical characteristics.

[0148] Preferably, the method further comprises the steps of:

[0149] a2) Pushing the recovery solution containing the nucleic acid solution obtained in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing the nucleic acid solution.

[0150] Preferably, step a2) is repeated 0-3 times.

[0151] Preferably, the nucleic acid-containing solution in step a1) comprises any one of: DNB, circular DNA, fragmented DNA, and a sequencing complex containing a DNB template, a polymerase, and a sequencing primer.

[0152] Further preferably, the nucleic acid-containing solution in step a1) comprises: DNB.

[0153] Preferably, the nucleic acid-containing solution in step a1) further comprises: a loading buffer.

[0154] Preferably, the amount of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times or 30 times greater than the nucleic acid adsorption capacity of the sequencing tank of the sequencing slide (i.e., the number of nucleic acid adsorption sites in the sequencing tank).

[0155] Preferably, the pushing method in steps a1), and / or a2) comprises the step of pushing using a simulated liquid containing a nucleic acid solution.

[0156] Preferably, the pushing method in steps a1), and / or a2) comprises the following steps: pushing a simulation liquid containing a nucleic acid solution into the liquid inlet of the sequencing tank.

[0157] Preferably, the simulated liquid containing the nucleic acid solution is pushed in by a pipetting device.

[0158] Preferably, before the simulation liquid containing nucleic acid solution is pushed into the liquid inlet of the sequencing tank, the following step is further included: pushing gas into the liquid inlet of the sequencing tank, wherein the gas does not affect the performance of the nucleic acid in the nucleic acid solution.

[0159] Preferably, the gas is pushed in by a pipetting device.

[0160] Preferably, the simulated liquid containing nucleic acid solution comprises an organic solvent, polyethylene glycol, and a buffer.

[0161] Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile.

[0162] Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000.

[0163] Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer.

[0164] Preferably, the pH of the buffer solution is 4.2-5.0.

[0165] Preferably, the concentration of the buffer solution is 0.1-0.4M.

[0166] Preferably, the final concentration of the buffer in the simulated solution containing nucleic acid solution is 62-88% (v / v).

[0167] Preferably, the final concentration of the polyethylene glycol in the simulated solution containing nucleic acid solution is 1-4% (m / v).

[0168] Preferably, the final concentration of the organic solvent in the simulated liquid containing nucleic acid solution is 10-30% (v / v).

[0169] Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides.

[0170] Preferably, in steps a1), and / or a2), the pushed nucleic acid-containing solution or the primary recovery liquid of the nucleic acid-containing solution is recovered by a pipetting device.

[0171] Preferably, the pipetting devices are each independently selected from at least one of a pipette, a syringe, and a syringe pump.

[0172] Preferably, the rolling circle amplification time of the DNB is 20-120 min.

[0173] Preferably, the empty sequencing slots of the sequencing carrier in a2) are capable of adsorbing nucleic acids in the nucleic acid-containing solution having the specific physical characteristics.

[0174] Preferably, the specific physical characteristics mentioned in a1) and a2) may be the same or different.

[0175] Preferably, the specific physical features mentioned in the repeated 2) may be the same or different.

[0176] Preferably, the physical characteristic comprises at least one of size, molecular mass and charge.

[0177] A sixth aspect of the present invention provides a simulated solution containing nucleic acid, comprising an organic solvent, polyethylene glycol, and a buffer solution.

[0178] Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile.

[0179] Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000.

[0180] Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer.

[0181] Preferably, the pH of the buffer solution is 4.2-5.0.

[0182] Preferably, the concentration of the buffer solution is 0.1-0.4M.

[0183] Preferably, the final concentration of the buffer in the simulated solution containing nucleic acid solution is 62-88% (v / v).

[0184] Preferably, the final concentration of the polyethylene glycol in the simulated solution containing nucleic acid solution is 1-4% (m / v).

[0185] Preferably, the final concentration of the organic solvent in the simulated liquid containing nucleic acid solution is 10-30% (v / v).

[0186] The beneficial effects of the present invention are:

[0187] The present invention provides a method for recycling a nucleic acid-containing solution for on-machine sequencing. The method can fully utilize the high-density feature of the nucleic acid-containing solution for on-machine sequencing. The density of the nucleic acid-containing solution used once will not be significantly reduced. The recovered nucleic acid-containing solution has the ability to be loaded onto a slide for sequencing multiple times (for example, again or three times), thereby improving the utilization rate of the nucleic acid-containing solution, achieving the purpose of reusing the nucleic acid-containing solution, and avoiding the waste of manpower and material resources caused by repeated preparation of nucleic acids (libraries). When applied to sequencing, under the premise of the need for repeated testing, the preparation process of the library and the nucleic acid-containing solution can be skipped, and re-sequencing can be directly performed, thereby reducing the sequencing process, sample preparation time and sequencing cost. Since the speed of nucleic acid sequencing, especially DNBSEQ sequencing, is getting faster and faster, the sequencing quality can be evaluated in as short as half an hour (up to more than 3 hours) from the loading of nucleic acids (such as DNBs). Therefore, the recovered nucleic acid-containing solution (such as DNBs) can be avoided. Long storage time affects its quality; it can avoid insufficient sequencing data due to too small sample size for rare samples, improve the utilization rate of rare samples, and further improve the depth of sequencing; improve the loading efficiency of some special libraries, such as when the library concentration is low or the DNB concentration is low, or the interaction between nucleic acid (such as DNB) and sequencing carrier (chip) is weak, it often leads to low loading efficiency, thus affecting data output; the nucleic acid-containing solution (such as DNB) recovered by this scheme is used to load the sequencing carrier (chip) for the second time, which can effectively improve the problem of low loading efficiency.

[0188] The present invention provides a system for recycling a nucleic acid-containing solution for on-machine sequencing, which can realize the automation of recycling the nucleic acid-containing solution for on-machine sequencing. The system accurately controls the entry of the nucleic acid-containing solution / recovery liquid of the nucleic acid-containing solution and the simulation liquid of the nucleic acid-containing solution through a pipetting device (syringe pump), thereby reducing the volume reduction of the recovered nucleic acid-containing solution. When applied to sequencing, the system can be used in conjunction with a sequencing module comprising a sequencing device to realize the automation of multiple sequencing operations.

[0189] The present invention provides a method for screening nucleic acids in a nucleic acid-containing solution with specific physical characteristics. The method can be used to screen nucleic acids with different characteristics in the nucleic acid-containing solution through the same operation as recovering the nucleic acid-containing solution. For example, a nucleic acid-containing solution containing DNBs often contains DNBs with different copy numbers, sizes or other characteristics (such as molecular mass, charge, etc.). A slide with certain characteristics can be used to specifically adsorb specific DNBs (such as a slide with a small adsorption site area adsorbing DNBs with a smaller volume). The number of the aforementioned specific DNBs in the recovered DNB mixture will be reduced, thereby achieving the screening purpose.

[0190] The present invention provides a simulating solution containing a nucleic acid solution. The simulating solution containing a nucleic acid solution has minimal impact on the nucleic acid solution during the recovery process and has no negative impact on the nucleic acid loaded on the recovered sequencing tank. Thus, the recovered solution containing the nucleic acid solution has minimal impact on the sequencing results after multiple loadings.

[0191] After the nucleic acid solution (e.g., DNB) enters the slide and incubates for a period of time, a postload process is generally required immediately to fix the nucleic acid (e.g., DNB) morphology. Before the postload process is complete, the introduction of different reagents may significantly affect the unstable nucleic acid (e.g., DNB) itself due to the presence of viscosity and shear forces. An appropriate nucleic acid solution simulant can avoid negative effects on the surface of unstable nucleic acids (e.g., DNB) during the recovery process. By limiting the pH and / or concentration (ionic strength) of the buffer solution, the pH value and / or concentration of the nucleic acid solution-containing simulation liquid and the nucleic acid solution-containing / nucleic acid solution-containing recovery liquid are made close, thereby ensuring that the nucleic acids (e.g., DNBs) loaded onto the sequencing slot of the sequencing slide will not undergo morphological changes over a long period of time. The organic solvent in the nucleic acid solution-containing simulation liquid and the alcohol in the polyethylene glycol can increase the rigidity of the nucleic acid solution-containing simulation liquid, making it less likely to generate bubbles when pushed in, and minimizing mixing between the nucleic acid solution-containing / nucleic acid solution-containing recovery liquid and the simulation liquid during contact, thereby avoiding excessive dilution of the nucleic acid solution to be recovered by the nucleic acid solution-containing simulation liquid. In addition, the nucleic acid solution-containing simulation liquid with the added alcohol component has a slight water absorption capacity, which can maintain the compact morphology of the nucleic acids (e.g., DNBs) on the sequencing slide. BRIEF DESCRIPTION OF THE DRAWINGS

[0192] FIG1 is a schematic diagram of obtaining DNA nanospheres by rolling circle amplification.

[0193] Figure 2 is a schematic diagram of the DNB loading process.

[0194] FIG3 is a schematic diagram of a conventional DNB loading process.

[0195] FIG4 is a schematic diagram of the present invention wherein the DNB mixture is loaded onto a sequencing slide, the DNB mixture is recovered, and the recovered DNB solution is loaded onto a sequencing slide.

[0196] FIG5 is a diagram showing the sequencing results of the recovered solution of the DNB mixture with rolling circle amplification times of 25 min and 120 min in Example 1.

[0197] FIG6 is a graph showing the sequencing results of the recovered liquid of the DNB mixture with a rolling circle amplification time of 120 min in Example 2 after one, two, and three recovery steps.

[0198] FIG7 is a diagram showing the sequencing results of the recovered liquid of the DNB mixture prepared from the human library in Example 3 after one and two recovery steps.

[0199] FIG8 is a diagram showing the sequencing results of the recovered liquid of the DNB mixture prepared from the single-cell RNA cDNA single-stranded circular DNA sequencing library in Example 4 after being recovered with different DNB simulation solutions. DETAILED DESCRIPTION

[0200] A first aspect of the present invention provides a method for recycling a nucleic acid-containing solution for sequencing, comprising the following steps:

[0201] a1) pushing out the nucleic acid-containing solution in the sequencing tank of the sequencing slide to obtain a recovery solution containing the nucleic acid solution (a primary recovery solution containing the nucleic acid solution);

[0202] a2) Pushing the recovery solution containing the nucleic acid solution (primary recovery solution containing the nucleic acid solution) obtained in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing the nucleic acid solution (primary recovery solution containing the nucleic acid solution).

[0203] Preferably, the nucleic acid-containing solution in step a1) comprises: DNA nanoballs (DNBs) (for example, when using a sequencing platform manufactured by MGI (for example, T series, G series, E series, etc.)), circular DNA (for example, when using a sequencing platform of Element Biosciences), fragmented DNA (for example, when using a sequencing platform of Illumina, Helicos Biosciences, etc.), a sequencing complex containing a DNA nanoball (DNB) template (the DNA nanoball template is preferably amplified from a dumbbell library), a polymerase, and a sequencing primer (for example, when using a sequencing platform of Pacific Biosciences, etc.).

[0204] Preferably, the nucleic acid-containing solution in step a1) (preferably when the nucleic acid-containing solution in step a1) contains DNB) further comprises: a loading buffer; wherein the loading buffer can be adjusted according to different sequencing platforms.

[0205] Preferably, the amount of nucleic acids (e.g., DNBs, circular DNA, fragmented DNA, DNA nanoball templates in sequencing complexes) in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times, or 30 times greater than the nucleic acid adsorption capacity of the sequencing tank of the sequencing slide (i.e., the number of nucleic acid adsorption sites in the sequencing tank).

[0206] Preferably, the nucleic acid-containing solution in step a1) further comprises a step of incubating in the sequencing tank before being ejected.

[0207] Preferably, before incubating in the sequencing tank, the method further comprises pushing a nucleic acid-containing solution into the sequencing tank.

[0208] Preferably, the loading buffer (preferably when the nucleic acid-containing solution in step a1) contains DNB) comprises at least one of citrate buffer, MES buffer solution, Tris hydrochloride buffer, acetate buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer; and further comprises at least one of citric acid-potassium citrate buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium citrate, and acetic acid-sodium acetate buffer.

[0209] Preferably, the pH of the loading buffer (preferably when the nucleic acid-containing solution in step a1) contains DNB) is 4.2-5.0; further 4.5-4.7.

[0210] Preferably, the concentration of the loading buffer (preferably when the nucleic acid-containing solution in step a1) contains DNB) is 0.1-0.4M; further 0.15-0.3M.

[0211] Preferably, step a2) is repeated 0-3 times; further preferably, step 2) is repeated 0-2 times; even further preferably, step 2) is repeated 0-1 times.

[0212] Preferably, when step a2) is repeated once, the method further comprises the following step: a3) pushing the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution) obtained by pushing out the recovery liquid containing nucleic acid solution (primary recovery liquid containing nucleic acid solution) in the previous step into an empty sequencing slot of the sequencing slide, incubating, and then pushing out the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution).

[0213] Preferably, when step a2) is repeated twice, the method further comprises the following steps: a3) pushing the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution) obtained by pushing out the recovery liquid containing nucleic acid solution (primary recovery liquid containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution); a4) pushing the recovery liquid containing nucleic acid solution (tertiary recovery liquid containing nucleic acid solution) obtained by pushing out the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery liquid containing nucleic acid solution (tertiary recovery liquid containing nucleic acid solution).

[0214] Preferably, when step a2) is repeated three times, the method further comprises the following steps: a3) pushing the recovery solution containing nucleic acid solution (secondary recovery solution containing nucleic acid solution) obtained by pushing out the recovery solution containing nucleic acid solution (primary recovery solution containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing nucleic acid solution (secondary recovery solution containing nucleic acid solution); a4) pushing the recovery solution containing nucleic acid solution (tertiary recovery solution containing nucleic acid solution) obtained by pushing out the recovery solution containing nucleic acid solution (secondary recovery solution containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing nucleic acid solution (tertiary recovery solution containing nucleic acid solution); a5) pushing the recovery solution containing nucleic acid solution (quaternary recovery solution containing nucleic acid solution) obtained by pushing out the recovery solution containing nucleic acid solution (tertiary recovery solution containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing nucleic acid solution (quaternary recovery solution containing nucleic acid solution).

[0215] Preferably, the method of pushing in steps a1), a2), a3), a4), and / or a5) comprises the step of pushing using a simulated liquid containing a nucleic acid solution; wherein the simulated liquid containing a nucleic acid solution can be adjusted according to different sequencing platforms.

[0216] Further preferably, the pushing method in steps a1), a2), a3), a4), and / or a5) comprises the following steps: pushing a simulation liquid containing a nucleic acid solution into the liquid inlet of the sequencing tank.

[0217] Preferably, the simulated liquid containing the nucleic acid solution is pushed in by a pipetting device.

[0218] Preferably, the volume of the simulated liquid containing nucleic acid solution is 0.5-1 times of the nucleic acid solution or the recovery liquid containing nucleic acid solution (such as the primary recovery liquid of the nucleic acid solution in a2), the secondary recovery liquid of the nucleic acid solution in a3), the tertiary recovery liquid of the nucleic acid solution in a4), or the fourth recovery liquid of the nucleic acid solution in a5); further 0.8-0.95 times.

[0219] Preferably, during the process of pushing in the simulated liquid containing nucleic acid solution, a small amount (eg, 0-5 μL, excluding 0; 0.5-3 μL; or 1-3 μL) of the simulated liquid containing nucleic acid solution is retained in the pipetting device to avoid pushing in all of it and causing bubbles.

[0220] Preferably, during the process of pushing the nucleic acid-containing solution in step a1), a small amount (for example, 0-5 μL, excluding 0; 0.5-3 μL or 1-3 μL) of the nucleic acid-containing solution is retained in the pipetting device to avoid pushing all the solution in and causing bubbles to be generated (although a small amount of the nucleic acid-containing solution is retained in the pipetting device, the pushed nucleic acid-containing solution can fill the sequencing tank).

[0221] Preferably, during the process of pushing the recovery liquid containing the nucleic acid solution in steps a2), a3), a4), and / or a5) (for example, pushing the primary recovery liquid containing the nucleic acid solution in a2), pushing the secondary recovery liquid containing the nucleic acid solution in a3), pushing the tertiary recovery liquid containing the nucleic acid solution in a4), and pushing the fourth recovery liquid containing the nucleic acid solution in a5), a small amount (for example, 0-3 μL, excluding 0; 0.5-2 μL; about 1 μL; or 1-1.5 μL) of the recovery liquid containing the nucleic acid solution (for example, pushing the primary recovery liquid containing the nucleic acid solution in a2), pushing the secondary recovery liquid containing the nucleic acid solution in a3), pushing the tertiary recovery liquid containing the nucleic acid solution in a4, and pushing the fourth recovery liquid containing the nucleic acid solution in a5) is retained in the pipetting device, so as to avoid pushing all the liquid in and causing bubbles to be generated (although a small amount of the recovery liquid containing the nucleic acid solution is retained in the pipetting device, the pushed recovery liquid containing the nucleic acid solution can fill the sequencing tank).

[0222] Preferably, the amount of recovery liquid containing nucleic acid solution (such as the primary recovery liquid containing nucleic acid solution, the secondary recovery liquid containing nucleic acid solution, the tertiary recovery liquid containing nucleic acid solution, and the quaternary recovery liquid containing nucleic acid solution) before being pushed into the empty sequencing slot of the sequencing carrier meets the needs of the next loading, and can thus fill the sequencing slot (if the recovery amount of a single sequencing slot cannot meet the needs of the next reloading, it is necessary to combine and collect recovery liquid containing nucleic acid solution to fill the volume of the sequencing slot before reloading): due to the inevitable volume loss during the recovery process, the recovery rate of the recovery liquid containing nucleic acid solution is between 50% and 90%, depending on the proportion of the simulated liquid containing nucleic acid solution in the volume of the sequencing slot. If the volume of the recovered nucleic acid solution is insufficient for reloading, the nucleic acid solution can be collected from multiple sequencing slots to be recovered until the volume is sufficient before loading into a new sequencing slot for sequencing.

[0223] Preferably, before the simulated liquid containing the nucleic acid solution is pushed into the liquid inlet of the sequencing tank, the following steps are also included: pushing a gas into the liquid inlet of the sequencing tank, wherein the gas does not affect the performance of the nucleic acid in the nucleic acid-containing solution, such as air; by pushing the gas before the simulated liquid containing the nucleic acid solution is pushed into the liquid inlet of the sequencing tank, an isolated gas column is formed to reduce the dilution of the nucleic acid-containing solution or the nucleic acid-containing solution recovery liquid (such as the primary recovery liquid of the nucleic acid-containing solution in a2), the secondary recovery liquid of the nucleic acid-containing solution in a3), the tertiary recovery liquid of the nucleic acid-containing solution in a4), or the quaternary recovery liquid of the nucleic acid-containing solution in a5) by the simulated liquid containing the nucleic acid solution.

[0224] Preferably, the gas is pushed in by a pipetting device.

[0225] Preferably, the volume of the gas is 0-5 μL; further preferably 2-3 μL.

[0226] Preferably, the sequencing slides in a1), a2), a3), a4), and a5) can be the same sequencing slide or different sequencing slides.

[0227] Preferably, the incubation time in a1), a2), a3), a4), and / or a5) is 5-60 min; further 10-30 min.

[0228] Preferably, in a1), a2), a3), a4), and / or a5), the pushed nucleic acid-containing solution or the recovery liquid containing the nucleic acid solution is recovered by a pipetting device (for example: in a1), the pushed nucleic acid-containing solution is recovered by a pipetting device to obtain a recovery liquid containing the nucleic acid solution (a primary recovery liquid containing the nucleic acid solution); in a2), the pushed nucleic acid-containing solution recovery liquid (a primary recovery liquid containing the nucleic acid solution) is recovered by a pipetting device to obtain a recovery liquid containing the nucleic acid solution (a secondary recovery liquid containing the nucleic acid solution); in a3), the pushed nucleic acid-containing solution recovery liquid (a secondary recovery liquid containing the nucleic acid solution) is recovered by a pipetting device to obtain a recovery liquid containing the nucleic acid solution (a tertiary recovery liquid containing the nucleic acid solution); in a4), the pushed nucleic acid-containing solution recovery liquid (a tertiary recovery liquid containing the nucleic acid solution) is recovered by a pipetting device to obtain a recovery liquid containing the nucleic acid solution (a quaternary recovery liquid containing the nucleic acid solution); in a5), the pushed nucleic acid-containing solution recovery liquid is recovered by a pipetting device).

[0229] Preferably, in a1), a2), a3), a4), and / or a5), the pushed nucleic acid-containing solution or the recovered liquid containing the nucleic acid solution is recovered at the liquid outlet of the sequencing tank by a pipetting device (for example: in a1), the pushed nucleic acid-containing solution is recovered at the liquid outlet of the sequencing tank by a pipetting device, in a2), the pushed primary recovered liquid containing the nucleic acid solution is recovered at the liquid outlet of the sequencing tank by a pipetting device, in a3), the pushed secondary recovered liquid containing the nucleic acid solution is recovered at the liquid outlet of the sequencing tank by a pipetting device, in a4), the pushed tertiary recovered liquid containing the nucleic acid solution is recovered at the liquid outlet of the sequencing tank by a pipetting device, and in a5), the pushed quaternary recovered liquid containing the nucleic acid solution is recovered at the liquid outlet of the sequencing tank by a pipetting device).

[0230] Preferably, during the process of recovering the pushed nucleic acid-containing solution or the recovered liquid of the nucleic acid-containing solution at the liquid outlet of the sequencing tank, the pipette tip of the pipetting device is not inserted into the small hole of the liquid outlet, thereby avoiding the formation of a cavity in the recovered liquid.

[0231] Preferably, the above-mentioned pipetting devices are each independently selected from at least one of a pipette, a syringe, and a syringe pump.

[0232] Preferably, the nucleic acid solution-containing simulating liquid (preferably a DNB simulating liquid) comprises an organic solvent, polyethylene glycol, and a buffer.

[0233] Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile; further is isopropanol.

[0234] Preferably, the organic solvent is an anhydrous organic solvent.

[0235] Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000; further PEG4000.

[0236] The organic solvent of the nucleic acid solution-containing simulating liquid and the alcohol in polyethylene glycol can increase the rigidity of the nucleic acid solution-containing simulating liquid, making it less likely to generate bubbles when pushed in. This can minimize mixing of the nucleic acid solution / nucleic acid solution-containing recovery liquid with the simulating liquid when in contact, thereby avoiding excessive dilution of the nucleic acid solution to be recovered by the nucleic acid solution-containing simulating liquid. In addition, the nucleic acid solution-containing simulating liquid with added alcohol components has a slight water absorption capacity, which can maintain the compact form of the nucleic acid (preferably DNB) in the nucleic acid solution on the sequencing slide.

[0237] Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer; and further comprises at least one of a citric acid-potassium citrate buffer, a disodium hydrogen phosphate-citric acid buffer, a citric acid-sodium citrate, and an acetic acid-sodium acetate buffer.

[0238] Preferably, the pH of the buffer solution is 4.2-5.0; further 4.5-4.7, further 4.6.

[0239] Preferably, the concentration of the buffer solution is 0.1-0.4M; further 0.15-0.3M.

[0240] Preferably, the final concentration of the buffer in the simulated solution containing nucleic acid solution is 62-88% (v / v); further 70-85% (v / v); further 80% (v / v).

[0241] By limiting the pH and / or concentration (ionic strength) and dosage of the buffer solution, the pH value and / or concentration of the simulated solution containing nucleic acid and the recovery solution containing nucleic acid / nucleic acid solution can be close, thereby ensuring that the nucleic acid (preferably DNB) in the nucleic acid-containing solution loaded on the sequencing tank of the sequencing carrier will not undergo morphological changes over a long period of time.

[0242] Preferably, the final concentration of the polyethylene glycol in the simulated solution containing nucleic acid solution is 1-4% (m / v); further 2-3% (m / v); further 2%.

[0243] Preferably, the final concentration of the organic solvent in the simulated solution containing nucleic acid solution is 10-30% (v / v); further 15-25%; further 16%.

[0244] Further preferably, the nucleic acid-containing solution comprises: DNB.

[0245] Preferably, the DNB is produced by Rolling Circle Amplification (RCA).

[0246] Preferably, the rolling circle amplification time of the DNB is 20-120 min; further 25-120 min.

[0247] Preferably, the rolling circle amplification (RCA) is a process of massively replicating DNA using single-stranded circular DNA as a template.

[0248] Preferably, the method for preparing the single-stranded circular DNA is a conventional method in the art, that is, circularizing a linear nucleic acid library.

[0249] Preferably, the method for constructing the linear nucleic acid library can be any method for constructing a linear nucleic acid library, including but not limited to the method for constructing a linear nucleic acid library mentioned in the prior art.

[0250] Preferably, the nucleic acid starting material for constructing the linear nucleic acid library can be any suitable DNA, RNA, or a complex of DNA and RNA, particularly DNA, and more particularly genomic DNA. The source of the nucleic acid is not limited and can be any biological source, such as nucleic acids from animals, plants, microorganisms, etc., particularly mammalian sources, especially human nucleic acids, and most preferably human genomic DNA.

[0251] More specifically,

[0252] The nucleic acid can be isolated from a biological sample obtained from an individual (e.g., a test individual). The individual can be any living or non-living organism, including but not limited to humans, non-human animals, plants, bacteria, fungi, protozoa, or pathogens.

[0253] Nucleic acids can be isolated or obtained from any type of suitable biological sample. Nucleic acids can be isolated or obtained from a single cell, a plurality of cells (e.g., cultured cells), a cell culture medium, a conditioned medium, a tissue, an organ, or an organism (e.g., bacteria, yeast, etc.).

[0254] Nucleic acids can be isolated or obtained from existing organisms or animals. In some cases, nucleic acids can be isolated or obtained from extinct (or "ancient") organisms or animals (e.g., extinct mammals, extinct mammals from the genus Homo, paleontological fossils). In some cases, nucleic acids can be obtained as part of a diagnostic assay.

[0255] In some cases, nucleic acids can be isolated or obtained from forensic samples or specimens. Forensic samples or specimens can include any biological material containing nucleic acids. For example, forensic samples or specimens can include blood, semen, hair, skin, sweat, saliva, decomposed tissue, bone, nail scrapings, licked stamps / envelopes, sluff, contact DNA, razor residue, etc. The specimen can be formalin-fixed tissue and / or paraffin-embedded tissue.

[0256] A biological sample can be any sample isolated or obtained from an individual or part thereof (e.g., a human individual, a pregnant female, a cancer patient, a patient suffering from an infection or infectious disease, a transplant recipient, a fetus, a tumor, an infected organ or tissue, a transplanted organ or tissue, a microbiome). In some embodiments, the biological sample is a cervical swab from an individual. The liquid sample or tissue sample from which nucleic acid is extracted can be acellular (e.g., free of cells). In some embodiments, the biological sample can contain cellular components or cell remnants. In some embodiments, the biological sample can include fetal cells or cancer cells.

[0257] Biological sample can be a liquid sample.Liquid sample can include extracellular nucleic acid (for example, circulating cell-free DNA).The example of liquid sample includes but is not limited to blood or blood products (such as serum, plasma, etc.), urine, cerebrospinal fluid, saliva, sputum, biopsy sample (for example, for detecting liquid biopsy of cancer), above-mentioned liquid sample, analog or its combination.In certain embodiments, biological sample is liquid biopsy, it generally refers to the assessment of the presence, absence, progression or alleviation of the liquid sample from individual about disease (for example, cancer).Liquid biopsy can be used in combination with the biopsy (for example, tumor biopsy) sold or used as its substitute.In some cases, extracellular nucleic acid is analyzed in liquid biopsy.

[0258] The biological sample can be a tumor nucleic acid sample (ie, a nucleic acid sample isolated from a tumor).

[0259] The second aspect of the present invention provides a system for recycling nucleic acid-containing solutions for on-machine sequencing, wherein the system is used to perform the method of the first aspect of the present invention.

[0260] Preferably, the system comprises:

[0261] A pushing module, which is used for pushing the nucleic acid-containing solution, the nucleic acid-containing solution simulation solution, the nucleic acid-containing solution recovery solution (such as the primary recovery solution containing nucleic acid solution in a2), the secondary recovery solution containing nucleic acid solution in a3), the tertiary recovery solution containing nucleic acid solution in a4), or the quaternary recovery solution containing nucleic acid solution in a5), and / or gas; and

[0262] A recovery module is used to recover the pushed nucleic acid-containing solution and / or the recovery liquid of the nucleic acid-containing solution (for example, the primary recovery liquid of the nucleic acid-containing solution in a2), the secondary recovery liquid of the nucleic acid-containing solution in a3), the tertiary recovery liquid of the nucleic acid-containing solution in a4), or the quaternary recovery liquid of the nucleic acid-containing solution in a5).

[0263] Preferably, the pushing module comprises a pipetting device.

[0264] Preferably, the pushing module further comprises a storage container.

[0265] Preferably, the storage container is used for storing a nucleic acid-containing solution, a simulated liquid containing a nucleic acid solution, and / or a gas.

[0266] Preferably, the recovery module comprises a pipetting device.

[0267] Preferably, the recovery module further comprises a recovery container (such as a separate recovery cabin).

[0268] Preferably, the recovery container is used for storing the recovery liquid containing the nucleic acid solution (for example: the primary recovery liquid containing the nucleic acid solution in a2), the secondary recovery liquid containing the nucleic acid solution in a3), the tertiary recovery liquid containing the nucleic acid solution in a4), or the fourth recovery liquid containing the nucleic acid solution in a5).

[0269] Preferably, the system is an automated system.

[0270] Preferably, the device in the pushing module and / or the recovery module is an automated device.

[0271] Preferably, the pipetting device comprises a syringe pump.

[0272] A third aspect of the present invention provides a sequencing method, comprising: the steps of the method for recycling the nucleic acid-containing solution for sequencing on a machine according to the first aspect of the present invention;

[0273] The method further comprises the step of sequencing the nucleic acid in the nucleic acid solution in the sequencing slot of the sequencing slide.

[0274] The fourth aspect of the present invention provides a sequencing system, which is used to perform the method of the third aspect of the present invention.

[0275] Preferably, the system comprises:

[0276] A pushing module, which is used for pushing the nucleic acid-containing solution, the nucleic acid-containing solution simulation solution, the nucleic acid-containing solution recovery solution (such as the primary recovery solution containing nucleic acid solution in a2), the secondary recovery solution containing nucleic acid solution in a3), the tertiary recovery solution containing nucleic acid solution in a4), or the quaternary recovery solution containing nucleic acid solution in a5), and / or gas; and

[0277] a recovery module for recovering the pushed nucleic acid-containing solution and / or the recovery solution of the nucleic acid-containing solution (e.g., the primary recovery solution of the nucleic acid-containing solution in a2), the secondary recovery solution of the nucleic acid-containing solution in a3), the tertiary recovery solution of the nucleic acid-containing solution in a4), or the quaternary recovery solution of the nucleic acid-containing solution in a5); and

[0278] The sequencing module is used to sequence the nucleic acid in the nucleic acid solution in the sequencing slot of the sequencing carrier.

[0279] Preferably, the pushing module comprises a pipetting device.

[0280] Preferably, the pushing module further comprises a storage container.

[0281] Preferably, the storage container is used for storing a nucleic acid-containing solution, a simulated liquid containing a nucleic acid solution, and / or a gas.

[0282] Preferably, the recovery module comprises a pipetting device.

[0283] Preferably, the recovery module further comprises a recovery container (such as a separate recovery cabin).

[0284] Preferably, the recovery container is used for storing the recovery liquid containing the nucleic acid solution (for example: the primary recovery liquid containing the nucleic acid solution in a2), the secondary recovery liquid containing the nucleic acid solution in a3), the tertiary recovery liquid containing the nucleic acid solution in a4), or the fourth recovery liquid containing the nucleic acid solution in a5).

[0285] Preferably, the sequencing module comprises a sequencing device.

[0286] Preferably, the system is an automated system.

[0287] Preferably, the devices in the pushing module, the recovery module, and / or the sequencing module are automated devices.

[0288] Preferably, the pipetting device comprises a syringe pump.

[0289] A fifth aspect of the present invention provides a method for screening nucleic acids (preferably screening DNBs) in a nucleic acid-containing solution having specific physical characteristics, comprising step a1) of the method for recycling a nucleic acid-containing solution for sequencing according to the first aspect of the present invention;

[0290] The sequencing slot of the sequencing carrier is capable of adsorbing nucleic acids in the nucleic acid-containing solution having the specific physical characteristics (preferably, the sequencing slot of the sequencing carrier is capable of adsorbing DNBs having the specific physical characteristics).

[0291] Preferably, the method further comprises step a2) of the method for recycling the nucleic acid-containing solution for sequencing on a machine according to the first aspect of the present invention.

[0292] Preferably, step a2) is repeated 0-3 times; further preferably, step 2) is repeated 0-2 times; even further preferably, step 2) is repeated 0-1 times.

[0293] Preferably, when step a2) is repeated once, the method further comprises the following step: a3) pushing the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution) obtained by pushing out the recovery liquid containing nucleic acid solution (primary recovery liquid containing nucleic acid solution) in the previous step into an empty sequencing slot of the sequencing slide, incubating, and then pushing out the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution).

[0294] Preferably, when step a2) is repeated twice, the method further comprises the following steps: a3) pushing the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution) obtained by pushing out the recovery liquid containing nucleic acid solution (primary recovery liquid containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution); a4) pushing the recovery liquid containing nucleic acid solution (tertiary recovery liquid containing nucleic acid solution) obtained by pushing out the recovery liquid containing nucleic acid solution (secondary recovery liquid containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery liquid containing nucleic acid solution (tertiary recovery liquid containing nucleic acid solution).

[0295] Preferably, when step a2) is repeated three times, the method further comprises the following steps: a3) pushing the recovery solution containing nucleic acid solution (secondary recovery solution containing nucleic acid solution) obtained by pushing out the recovery solution containing nucleic acid solution (primary recovery solution containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing nucleic acid solution (secondary recovery solution containing nucleic acid solution); a4) pushing the recovery solution containing nucleic acid solution (tertiary recovery solution containing nucleic acid solution) obtained by pushing out the recovery solution containing nucleic acid solution (secondary recovery solution containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing nucleic acid solution (tertiary recovery solution containing nucleic acid solution); a5) pushing the recovery solution containing nucleic acid solution (quaternary recovery solution containing nucleic acid solution) obtained by pushing out the recovery solution containing nucleic acid solution (tertiary recovery solution containing nucleic acid solution) in the previous step into an empty sequencing slot of a sequencing slide, incubating, and then pushing out the recovery solution containing nucleic acid solution (quaternary recovery solution containing nucleic acid solution).

[0296] Preferably, the empty sequencing slots of the sequencing slides in a2), a3), a4), and / or a5) are capable of adsorbing nucleic acids in the nucleic acid-containing solution having the specific physical characteristics.

[0297] Preferably, the specific physical characteristics in a1), a2), a3), a4) and a5) may be the same or different.

[0298] Preferably, the physical characteristic comprises at least one of size, molecular mass, and charge; further comprises size.

[0299] The further limitations on any technical features in steps a1), a2), a3), a4), and / or a5) are the same as those in the first aspect of the present invention.

[0300] The sixth aspect of the present invention provides a simulated solution containing nucleic acid solution (preferably a DNB simulated solution), wherein the simulated solution containing nucleic acid solution is the simulated solution containing nucleic acid solution in the first aspect of the present invention.

[0301] The present invention is further described in detail below through specific examples.

[0302] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0303] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0304] The sequencing slides used in this example (Cat. No.: 1000008403), standard library reagent V3.0 (Cat. No.: 1000005033), Make DNB enzyme MIX Ⅰ (Cat. No.: 1000005297) and Make DNB enzyme Ⅱ (Cat. No.: 1000004803), stop run The buffer (catalog number: 1000005054) and other reagents were all from the MGISEQ-2000 standard PE100 sequencing reagent set (catalog number: 1000012536), provided by Wuhan MGI. The anhydrous disodium hydrogen phosphate (catalog number A501727) and citric acid monohydrate (catalog number: A502123) used were purchased from Sangon Biotech Co., Ltd., and isopropanol (catalog number I112011-500 mL) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. 50% (m / v) PEG4000 solution was provided by Qingdao MGI.

[0305] When sequencing using the MGISEQ-2000RS sequencer, the MGISEQ-2000RS sequencer is produced and provided by Wuhan MGI Intelligent Manufacturing. The sequencing primers, sequencing reagents, and sequencing slides used are all from the MGISEQ-2000 standard PE100 sequencing reagent set, provided by Wuhan MGI Intelligent Manufacturing.

[0306] Example 1: Recovery and resequencing of DNBs with different rolling amplification times

[0307] 1. Prepare DNB mixture using Ecoli library standard sample V3

[0308] (1) Prepare a DNB mixture using Ecoli library standard sample V3 with a rolling circle amplification time of 25 minutes

[0309] 1) Using eight 0.2 mL PCR tubes, prepare the reaction mixture on ice as described in Table 1. Mix the resulting mixture on a rotary shaker, centrifuge briefly, and place in a PCR instrument with the following settings: 95°C for 1 minute, 65°C for 1 minute, 40°C for 1 minute, and a 4°C hold. Set the heated lid to 95°C.

[0310] 2) After the PCR instrument reaches 4°C, remove the PCR tube, centrifuge for 5 seconds, and place on ice. Add 40 μL of DNB Polymerase Mix I and 1.6 μL of DNB Polymerase Mix II (LC) to the PCR tube while still on ice.

[0311] 3) Mix the resulting reaction mixture using a vortex shaker and centrifuge for 5 seconds. Immediately place the reaction mixture in a PCR instrument and set it to 30°C for 25 minutes and hold at 4°C. Set the heated lid to 35°C.

[0312] When the temperature reaches 4°C, immediately add 20 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly with a wide-mouth pipette tip 5-8 times to obtain the DNB solution, which can be stored in a 4°C refrigerator until used.

[0313] Before DNB loading, take the DNB solution and add DNB loading buffer II with 1 / 3 volume of the DNB solution. Slowly mix 5-8 times with a wide-mouth pipette tip to obtain the DNB mixture, which must be prepared and used immediately.

[0314] Table 1 DNB preparation reaction system

[0315] (2) Prepare a DNB mixture with a rolling circle amplification time of 120 min using Ecoli library standard sample V3

[0316] The preparation method is the same as (1), except that the step 3) is changed from "setting to 30°C for 25 min" to "setting to 30°C for 120 min".

[0317] 2. Preparation of DNB Simulation Solution

[0318] (1) Weigh 2.84 g of anhydrous disodium hydrogen phosphate and dilute to 100 mL with ultrapure water to prepare a 0.2 mol / L Na2HPO4 solution; weigh 2.1 g of citric acid monohydrate and dilute to 100 mL with ultrapure water to prepare a 0.1 mol / L citric acid solution;

[0319] (2) Use 4.675 mL of 0.2 mol / L Na2HPO4 solution and 5.325 mL of 0.1 mol / L citric acid solution to prepare 10 mL of sodium dihydrogen phosphate-citric acid buffer with a pH of 4.6;

[0320] (3) 1 mL of DNB simulation solution was prepared by mixing 800 μL of sodium hydrogen phosphate-citrate buffer (pH 4.6), 160 μL of isopropanol (final concentration 16%, v / v), and 40 μL of 50% (m / v) PEG4000 solution (final concentration 2%, m / v). The mixture was thoroughly mixed and centrifuged, and then placed at 4°C for use.

[0321] 3.DNB loading sequencing slide and recovery sequencing

[0322] Two sequencing slides (both MGISEQ-2000RS sequencing slides with catalog number 1000008403, each slide has four identical sequencing slots, designated lane 1, lane 2, lane 3, and lane 4) were used to sequence the DNB mixture prepared in step 1 with a rolling circle amplification time of 25 and 120 minutes, and the recovered DNB mixture, respectively, as follows:

[0323] (1) Use a 200 μL pipette tip to take 30 μL of the DNB mixture prepared in step 1 with a rolling circle amplification time of 25 min, align the tip of the pipette tip with the liquid inlet hole of lane 1 of the sequencing slide, and slowly push the DNB mixture into the pipette tip. Stop pushing when there is 1-3 μL of DNB mixture remaining in the pipette tip. At this time, the DNB solution covers the entire sequencing slot (for a sequencing slot of the MGISEQ2000 sequencing slide, the volume is about 26 μL, so even if there is 1-3 μL remaining in the pipette tip, the entire slide can be covered);

[0324] (2) Add the DNB mixture to lane 2 and lane 3 according to the method of step (1);

[0325] (3) After incubating the DNB mixture for 10 minutes, use a 200 μL pipette tip to draw 30 μL of the DNB simulated solution prepared in step 2, align the tip of the pipette tip with the liquid inlet holes of lane 1, lane 2, and lane 3, and slowly push the DNB simulated solution in. Stop pushing when 1-3 μL of simulated solution remains in the pipette tip (steps (2) and (3) are shown in Figure 3);

[0326] (4) Use a pipette to recycle the DNB mixture in the outlets of lanes 1, 2, and 3 to obtain DNB recovery solution. Use a 200 μL pipette tip to draw 30 μL of DNB recovery solution. Align the tip of the pipette tip with the small hole of the lane 4 inlet and slowly push it in until 1-3 μL remains in the pipette tip. Stop and incubate for 10 min.

[0327] (5) According to steps (1)-(4), the DNB mixture prepared in step 1 with a rolling circle amplification time of 120 min is loaded and recovered on lanes 1, 2, and 3 of another sequencing slide, and the DNB recovery solution is loaded on lane 4;

[0328] (6) Perform PE100+10 sequencing on the sequencing slide loaded with DNB obtained in steps (4) and (5) on the MGISEQ-2000 sequencer. For sequencing operations, refer to the MGISEQ-2000RS-High-throughput (Fast) Sequencing Reagent Set Instructions (SOP-013-B01-054-A7) of MGI Intelligent Manufacturing Co., Ltd.

[0329] The results are shown in Figure 5: Regardless of whether the DNB with a rolling circle amplification time of 25 minutes or the DNB with a rolling circle amplification time of 120 minutes, the overall performance of the recovered DNB is not worse than that of the normally loaded (initial loading) DNB. Even the overall sequencing performance (such as various indicators such as the output read length (TotalReads), overall Q30, effective data ratio (ESR)), etc.) is better than that of the normally loaded DNB, indicating that the recovered DNB has good sequencing performance, and the DNB mixture with an RCA time as short as 25 minutes and as long as 120 minutes has good recycling and utilization.

[0330] Example 2 DNB sequencing effects with different recovery times

[0331] 1. Prepare DNB mixture using Ecoli library standard sample V3

[0332] The method is the same as the method for preparing the DNB mixture with a rolling circle amplification time of 120 minutes in Example 1.

[0333] 2. Preparation of DNB Simulation Solution

[0334] The method is the same as the preparation method of the DNB simulated liquid in Example 1.

[0335] 3.DNB loading sequencing slide and recovery sequencing

[0336] A sequencing slide (MGISEQ-2000RS sequencing slide with catalog number 1000008403, each slide has four identical sequencing slots, designated lane 1, lane 2, lane 3, and lane 4) was used to sequence the DNB mixture prepared in step 1 with a rolling circle amplification time of 120 minutes and the recovered DNB mixture, as follows:

[0337] (1) First recovery of DNB mixture and loading lane 2: Use a 200 μL pipette to take 30 μL of DNB mixture, align the tip of the pipette with the liquid inlet hole, and slowly push the DNB mixture into the sequencing tank lane 1. Stop pushing when there is 1-3 μL of DNB mixture left in the pipette; After the DNB mixture is incubated for 10 minutes, use a 200 μL pipette to aspirate 30 μL of DNB simulation solution, align the tip of the pipette with the liquid inlet hole of lane 1, and slowly push the DNB simulation solution in step 2 into the pipette. Stop pushing when there is 1-3 μL of simulation solution left in the pipette; Recover the DNB mixture in the liquid outlet of lane 1, and use a 200 μL pipette to aspirate all the DNB recovery solution, align the tip of the pipette with the liquid inlet hole of lane 2, and slowly push until there is about 1 μL left in the pipette, and incubate for 10 minutes (Figure 4);

[0338] (2) Secondary recovery of the DNB mixture and loading lane 3: After the DNB mixture on lane 2 has been incubated for 10 min, 30 μL of the DNB simulated solution from step 2 is aspirated with a 200 μL pipette tip, the tip of the pipette tip is aligned with the small hole of the lane 2 liquid inlet, and the DNB simulated solution is slowly pushed in. When 1-3 μL of simulated solution remains in the pipette tip, the DNB mixture at the lane 2 liquid outlet is recovered using a pipette, and all the DNB recovery solution is aspirated with a 200 μL pipette tip, the tip of the pipette tip is aligned with the small hole of the lane 3 liquid inlet, and the DNB simulated solution is slowly pushed in until about 1 μL remains in the pipette tip, and the mixture is incubated for 10 min.

[0339] (3) Recover DNBs three times and load lane 4: After the DNB mixture on lane 3 is incubated for 10 minutes, 30 μL of the DNB simulation solution in step 2 is aspirated with a 200 μL pipette tip, the tip of the pipette tip is aligned with the small hole of the lane 3 liquid inlet, and the DNB simulation solution is slowly pushed in. When 1-3 μL of simulation solution remains in the pipette tip, the pushing is stopped; the DNB mixture existing in the lane 3 liquid outlet is recovered, and all the DNB recovery solution is aspirated with a 200 μL pipette tip, the tip of the pipette tip is aligned with the small hole of the lane 4 liquid inlet, and the DNB recovery solution is slowly pushed in until about 1 μL remains in the pipette tip, and incubated for 10 minutes; that is, for different sequencing tanks: lane 1 is the original DNB mixture (DNB mixture loaded for the first time), lane 2 is the DNB mixture recovered once, lane 3 is the DNB mixture recovered twice, and lane 4 is the DNB mixture recovered three times;

[0340] (4) Perform PE100+10 sequencing on the sequencing slide loaded with DNB obtained in step (3) on the MGISEQ-2000 sequencer. For sequencing operations, refer to the MGISEQ-2000RS-High-throughput (Fast) Sequencing Reagent Set Instructions (SOP-013-B01-054-A7) of MGI Intelligent Manufacturing Company.

[0341] The results are shown in Figure 6: DNBs that were recycled once and twice were slightly inferior to DNBs that were normally loaded (initial loading) in terms of output read length (TotalReads) and overall Q30. The data volume of DNBs that were recycled three times decreased significantly, but the data was still usable. The overall trend is that the more times the recycling is done, the lower the data output is, which is a normal phenomenon. It is recommended to control the number of repeated recycling of DNBs to within 2 times during use to achieve relatively good sequencing results. It should be noted that the RCA time of 120min for DNBs is not the recommended RCA time. The recommended RCA time is 25min. The RCA time of 120min for DNBs is itself under relatively extreme conditions, so there may be some deviation in sequencing quality.

[0342] Example 3: Secondary recovery sequencing of DNBs prepared from human samples

[0343] In this example, nucleic acid samples were extracted from human blood using the MGIEasy Nucleic Acid Extraction Reagent (OP02-32) (Cat. No. 1000023774), and sequencing libraries were prepared using the MGIEasy Enzyme-Digested PCR-Free DNA Library Preparation Kit (16RXN) (Cat. No. 1000013454). DNBs were prepared from the prepared sequencing libraries and then subjected to secondary recovery sequencing to observe their performance, as follows:

[0344] 1. Prepare DNB mixture using human sequencing library samples

[0345] A DNB mixture was prepared according to (1) in step 1 of Example 1, and the RCA time was 25 min.

[0346] 2. Preparation of DNB Simulation Solution

[0347] The method is the same as the preparation method of the DNB simulation solution in Example 1 (DNB simulation solution 1). In addition, 300 μL of loading buffer I and 100 μL of loading buffer II in the sequencing kit are mixed to obtain DNB simulation solution 2 without adding PEG4000 and isopropanol.

[0348] 3.DNB loading sequencing slide and recovery sequencing

[0349] A sequencing slide (MGISEQ-2000RS sequencing slide with catalog number 1000008403, a single slide has four identical sequencing slots, designated lane 1, lane 2, lane 3, and lane 4) was used to sequence the DNB mixture prepared in step 1 and the recovered DNB mixture as follows:

[0350] (1) Loading lanes 1 and 2 with the DNB mixture: Use a 200 μL pipette tip to take 30 μL of the DNB mixture from step 1. Align the tip of the pipette tip with the small hole in the inlet of lane 1 and slowly push it in. Stop when 1-3 μL of DNB mixture remains in the pipette tip. Load lane 2 with DNB in ​​the same way. Incubate for 10 minutes.

[0351] (2) Recover the DNBs in lane 1 and lane 2: Use a 200 μL pipette tip to aspirate 30 μL of the DNB simulation solution 1 prepared in step 2, align the tip of the pipette tip with the small hole of the lane 1 liquid inlet, and slowly push it in. Stop pushing when there is 1-3 μL of simulation solution left in the pipette tip, and use a pipette to recover the DNBs pushed out of the lane 1 liquid outlet. Use a 200 μL pipette tip to take 30 μL of the DNB simulation solution 2 prepared in step 2 (mixed by a 3:1 volume ratio of loading buffer I and loading buffer II in the sequencing kit without adding PEG4000 and isopropanol), align the tip of the pipette tip with the small hole of the lane 2 liquid inlet, and slowly push it in. Stop pushing when there is 1-3 μL of DNB simulation solution left in the pipette tip, and use a pipette to recover the DNBs pushed out of the lane 2 liquid outlet.

[0352] (3) Load lane 3 with the DNB mixture recovered from lane 1: Use a 200 μL pipette tip to aspirate all the DNB recovery solution obtained from lane 1, align the tip of the pipette tip with the small hole of the lane 3 inlet, and slowly push it in until about 1 μL remains in the pipette tip, and incubate for 10 min;

[0353] (4) Secondary recovery of the DNB mixture and loading lane 4: Use a 200 μL pipette to draw 30 μL of the DNB simulated solution 1 in step 2, align the tip of the pipette with the small hole of the lane 3 liquid inlet, and slowly push the DNB simulated solution in. Stop pushing when there is 1-3 μL of DNB simulated solution left in the pipette; Use a pipette to recover the DNB mixture in the lane 3 liquid outlet, and use a 200 μL pipette to draw all the DNB recovery solution, align the tip of the pipette with the small hole of the lane 4 liquid inlet, and slowly push it in until there is about 1 μL left in the pipette, and incubate for 10 minutes;

[0354] (5) Perform PE100+10 sequencing on the sequencing slide loaded with DNB obtained in step (4) on the MGISEQ-2000 sequencer. For sequencing operations, refer to the MGISEQ-2000RS-High-throughput (Fast) Sequencing Reagent Set Instructions (SOP-013-B01-054-A7) of MGI Intelligent Manufacturing Company.

[0355] The results are shown in Figure 7 and Table 2: On the one hand, the use of the DNB simulation solution of the present invention for the push-out of the DNB mixture (treatment 1) has a better effect than the use of DNB simulation solution 2 prepared by mixing DNB loading buffers I and II in a ratio of 3:1 (treatment 2). The various sequencing indicators of the recovered sequencing tank itself are significantly higher, indicating that the DNB simulation solution with the addition of isopropanol and PEG4000 of the present invention is more suitable for pushing out DNBs, and the push-out operation has little effect on the DNBs loaded on the sequencing slide. On the other hand, the sequencing performance of the DNBs recovered once (treatment 3) and twice (treatment 4) did not decline, and various indicators such as the output read length (TotalReads), overall Q30, and effective data ratio (ESR) were significantly better than those of the recovered sequencing tank (treatment 1), indicating that the DNBs recovered once and twice have a sequencing performance that is not inferior to or even better than normal sequencing, and that the primary and secondary DNB recovery methods of the present invention have good applicability for human sample libraries.

[0356] Table 2 Sequencing results of the recovered liquid of the DNB mixture prepared from the human library after one and two recovery

[0357] Example 4: Using different DNB simulation solutions to recover DNBs from single-cell RNA cDNA samples and then sequence them

[0358] 1. Prepare single-cell RNA cDNA single-stranded circular DNA sequencing library from sample

[0359] Mouse (Mus musculus) brain cell nuclear suspension samples were provided by the Reagent R&D Department of Qingdao MGI Intelligent Manufacturing Technology Co., Ltd., and single-cell RNA cDNA single-stranded circular DNA sequencing libraries were subsequently prepared using the MGI C-series High-Throughput RNA Library Preparation Kit (Cat. No.: 940-000519-00).

[0360] 2. Preparation of DNB Mixture Using Single-Cell RNA cDNA Single-Stranded Circular DNA Sequencing Library

[0361] 1) Using eight 0.2 mL PCR tubes, prepare the reaction mixture on ice as described in Table 3. Mix the resulting mixture on a rotary shaker, centrifuge briefly, and place in a PCR instrument with the following settings: 95°C for 1 minute, 65°C for 1 minute, 40°C for 1 minute, and a 4°C hold. Set the heated lid to 95°C.

[0362] 2) After the PCR instrument reaches 4°C, remove the PCR tube, centrifuge for 5 seconds, and place on ice. Add 40 μL of DNB Polymerase Mix I and 1.6 μL of DNB Polymerase Mix II (LC) to the PCR tube while still on ice.

[0363] 3) Mix the resulting reaction mixture using a vortex shaker and centrifuge for 5 seconds. Immediately place the reaction mixture in a PCR instrument and set it to 30°C for 20 minutes and a 4°C hold temperature. Set the heated lid to 35°C.

[0364] When the temperature reaches 4°C, immediately add 20 μL of DNB stop buffer and mix thoroughly by pipetting up and down slowly with a wide-mouth pipette tip 5-8 times to obtain the DNB solution, which can be stored in a 4°C refrigerator until used.

[0365] Before DNB loading, take the DNB solution and add DNB loading buffer II with 1 / 3 volume of the DNB solution. Slowly mix 5-8 times with a wide-mouth pipette tip to obtain the single-cell RNA cDNA sample DNB mixture, which must be prepared and used immediately.

[0366] Table 3 DNB preparation reaction system 2

[0367] 3. Preparation of different DNB simulation solutions

[0368] a1) The method for preparing the DNB simulated solution is the same as that in Example 1, i.e., 800 μL of sodium hydrogen phosphate-citrate buffer (pH 4.6), 160 μL of isopropanol (final concentration 16%, v / v), and 40 μL of 50% (m / v) PEG4000 solution (final concentration 2%, m / v) are mixed to form 1 mL of the DNB simulated solution. After thorough mixing and centrifugation, the solution is stored at 4°C until use.

[0369] a2) The method for preparing the DNB simulating solution is the same as that in Example 1, except that only sodium hydrogen phosphate-citrate buffer with a pH of 4.6 is used, without adding isopropanol, PEG4000 or other substitutes, as the DNB simulating solution, and the solution is stored at 4° C. until use;

[0370] a3) The method for preparing the DNB simulated solution was the same as that in Example 1, except that 880 μL of sodium hydrogen phosphate-citrate buffer (pH 4.6), 100 μL of isopropanol (final concentration 10%, v / v), and 20 μL of 50% (m / v) PEG4000 solution (final concentration 1%, m / v) were mixed to form 1 mL of the DNB simulated solution. The mixture was thoroughly mixed, centrifuged, and stored at 4°C until use.

[0371] a4) The method for preparing the DNB simulated solution is the same as that in Example 1, except that 620 μL of sodium hydrogen phosphate-citrate buffer (pH 4.6), 300 μL of isopropanol (final concentration 30%, v / v), and 80 μL of 50% (m / v) PEG4000 solution (final concentration 4%, m / v) are mixed to form 1 mL of the DNB simulated solution. The mixture is thoroughly mixed, centrifuged, and stored at 4°C until use.

[0372] 4.DNB loading sequencing slide and recovery sequencing

[0373] Two sequencing slides (both MGISEQ-2000RS sequencing slides with catalog number 1000008403, each with a total of 8 sequencing slots, designated lane 1, lane 2, lane 3, lane 4, lane 5, lane 6, lane 7, and lane 8 in this example) were used. Sequencing was performed on the MGISEQ-2000 sequencer for 30 reads per strand (SE30) on the single-cell cDNA DNB mixture prepared in step 1 and the DNBs recovered using different DNB simulation solutions in step 3, as follows:

[0374] (1) Use a 200 μL pipette tip to take 30 μL of the DNB mixture prepared in step 1, align the tip of the pipette tip with the liquid inlet hole of lane 1 of the sequencing slide, and slowly push the DNB mixture into the pipette tip. Stop pushing when 1-3 μL remains in the pipette tip.

[0375] (2) According to the method of step (1), the DNB mixture is loaded into lane 2, lane 3, lane 5, and lane 7;

[0376] (3) After incubating the DNB mixture for 10 minutes, use a 200 μL pipette tip to draw 30 μL of the DNB simulated solution a2) prepared in step 3. Align the tip of the pipette tip with the small hole in the lane 2 inlet and slowly push the DNB simulated solution in. Stop pushing when 1-3 μL of simulated solution remains in the pipette tip. Recover the DNB mixture at the lane 2 outlet.

[0377] (4) Use a 200 μL pipette tip to draw 30 μL of the DNB simulated solution a1) prepared in step 3. Align the tip of the pipette tip with the small hole in the lane 3 liquid inlet and slowly push the DNB simulated solution in. Stop pushing when 1-3 μL of simulated solution remains in the pipette tip. Recover the DNB mixture in the lane 3 liquid outlet.

[0378] (5) Use a 200 μL pipette tip to draw 30 μL of the DNB solution (a3) ​​prepared in step 3. Align the tip of the pipette tip with the small hole in the lane 5 inlet and slowly push the DNB solution in. Stop pushing when 1-3 μL of solution remains in the pipette tip. Recover the DNB solution at the lane 5 outlet.

[0379] (6) Use a 200 μL pipette tip to draw 30 μL of the DNB solution (a4) prepared in step 3. Align the tip of the pipette tip with the small hole in the lane 7 inlet and slowly push the DNB solution in. Stop pushing when 1-3 μL of solution remains in the pipette tip. Recover the DNB solution at the lane 7 outlet.

[0380] (7) Loading of recovered DNBs: Using a 200 μL pipette tip, pipette the DNB mixture recovered from lane 3 into lane 4; pipette the DNB mixture recovered from lane 5 into lane 6; and pipette the DNB mixture recovered from lane 7 into lane 8. Stop when approximately 1 μL remains in the pipette tip.

[0381] (8) Perform SE30 sequencing on the two sequencing slides obtained in step (7) on the MGISEQ-2000 sequencer. For sequencing operations, refer to the MGISEQ-2000RS-High-throughput (Rapid) Sequencing Reagent Set Instructions (SOP-013-B01-054-A7) of MGI Intelligent Manufacturing Co., Ltd.

[0382] The results are shown in Figure 8 and Table 4. For the recommended range of the DNB simulation solution formulation of the present invention, when using the upper limit addition amount of isopropyl alcohol and PEG4000 (i.e., a4) simulation solution) or the lower limit addition amount of isopropyl alcohol and PEG4000 (i.e., a3) simulation solution), the sequencing performance of both the pushed-out sequencing tank and the recovered DNBs (such as the overall Q30, initial Q30, and the Q30 curve formed by the Q30 of each cycle) were significantly better than those of the recovered sequencing tank (Treatment 1), and were significantly better than those of the normally loaded DNBs. The most recommended DNB simulation solution formulation of the present invention, i.e., a1) simulation solution, achieved good performance in both the pushed-out sequencing tank and the recovered DNBs (such as the overall Q30, initial Q30, and the Q30 curve formed by the Q30 of each cycle). Therefore, DNB simulation solutions prepared by adjusting the formulation within the recommended range all exhibited relatively good DNB recovery effects. For the DNB simulation solution without the addition of isopropanol and PEG4000, the sequencing performance of the solution pushed out of the sequencing tank was at the worst level and inferior to that of the normally loaded DNB, indicating that it has a negative impact on the loaded DNB and is not suitable for direct use for DNB recovery. The addition of isopropanol and PEG4000 or their substitutes in the present invention is still required.

[0383] Table 4 Sequencing results of the recovered liquids of the DNB mixture prepared from the single-cell RNA cDNA single-stranded circular DNA sequencing library after being recovered with different DNB simulation solutions

[0384] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for recycling a nucleic acid-containing solution for on-machine sequencing, comprising the following steps: a1) Pushing out the nucleic acid-containing solution in the sequencing groove of the sequencing slide to obtain a recovered solution of the nucleic acid-containing solution; a2) Pushing the recovered solution of the nucleic acid-containing solution obtained in the previous step into the empty sequencing groove of the sequencing slide, incubating, and then pushing out the recovered solution of the nucleic acid-containing solution.

2. The method according to claim 1, wherein: Step a2) is repeated 0-3 times; Preferably, the nucleic acid-containing solution in step a1) comprises any one of DNB, circular DNA, fragment DNA, and a sequencing complex containing a DNB template, a polymerase, and a sequencing primer; Preferably, the nucleic acid-containing solution in step a1) further comprises: a loading buffer; Preferably, the amount of nucleic acid in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times, or more than 30 times the nucleic acid adsorption capacity of the sequencing groove of the sequencing slide.

3. The method according to claim 2, wherein: The pushing-out method in step a1) and / or a2) comprises a step of pushing out with a simulated solution of the nucleic acid-containing solution; Preferably, the pushing-out method in step a1) and / or a2) comprises the following steps: pushing a simulated solution of the nucleic acid-containing solution into the liquid inlet of the sequencing groove; Preferably, the simulated solution of the nucleic acid-containing solution is pushed in by a pipetting device; Preferably, before pushing a simulated solution of the nucleic acid-containing solution into the liquid inlet of the sequencing groove, the following steps are further included: pushing a gas into the liquid inlet of the sequencing groove, and the gas does not affect the performance of the nucleic acid in the nucleic acid-containing solution; Preferably, the gas is pushed in by a pipetting device; Preferably, the simulated solution of the nucleic acid-containing solution comprises an organic solvent, polyethylene glycol, and a buffer; Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile; Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000; Preferably, the buffer comprises at least one of citrate buffer, MES buffer solution, Tris hydrochloride buffer, acetate buffer, disodium hydrogen phosphate–citric acid buffer, and citric acid–sodium hydroxide–hydrochloric acid buffer; Preferably, the pH of the buffer is 4.2-5.0; Preferably, the concentration of the buffer is 0.1-0.4M; Preferably, the final concentration of the buffer in the simulated solution of the nucleic acid-containing solution is 62-88% (v / v); Preferably, the final concentration of the polyethylene glycol in the simulated solution of the nucleic acid-containing solution is 1-4% (m / v); Preferably, the final concentration of the organic solvent in the simulated solution of the nucleic acid-containing solution is 10-30% (v / v); Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides; Preferably, in a1) and / or a2), the pushed-out nucleic acid-containing solution or the recovered solution of the nucleic acid-containing solution is recovered by a pipetting device; Preferably, each of the pipetting devices is independently selected from at least one of a pipette, a syringe, and an injection pump; Preferably, the nucleic acid-containing solution in step a1) contains: DNB; Preferably, the time for rolling circle amplification of the DNB is 20 - 120 min.

4. A recycling system for a nucleic acid-containing solution for next-generation sequencing, the system being used to perform a method for recycling a nucleic acid-containing solution for next-generation sequencing, the method comprising the following steps: a1) Pushing out the nucleic acid-containing solution in the sequencing groove of the sequencing slide to obtain a recovered solution of the nucleic acid-containing solution; a2) Pushing the recovered solution of the nucleic acid-containing solution obtained in the previous step into the empty sequencing groove of the sequencing slide, incubating, and then pushing out the recovered solution of the nucleic acid-containing solution.

5. The system according to claim 4, wherein: Step a2) is repeated 0 - 3 times; Preferably, the nucleic acid-containing solution in step a1) contains any one of DNB, circular DNA, fragmented DNA, a sequencing complex containing a DNB template, a polymerase, and a sequencing primer; Preferably, the nucleic acid-containing solution in step a1) further contains: a loading buffer; Preferably, the quantity of nucleic acid in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times, or more than 30 times the nucleic acid adsorption capacity of the sequencing groove of the sequencing slide; Preferably, the pushing-out method in step a1) and / or a2) includes a step of pushing out with a simulated solution of the nucleic acid-containing solution; Preferably, the pushing-out method in step a1) and / or a2) includes the following steps: pushing a simulated solution of the nucleic acid-containing solution into the liquid inlet of the sequencing groove; Preferably, the simulated solution of the nucleic acid-containing solution is pushed in by a pipetting device; Preferably, before pushing a simulated solution of the nucleic acid-containing solution into the liquid inlet of the sequencing groove, the following step is further included: pushing a gas into the liquid inlet of the sequencing groove, the gas not affecting the performance of the nucleic acid in the nucleic acid-containing solution; Preferably, the gas is pushed in by a pipetting device; Preferably, the simulated solution of the nucleic acid-containing solution contains an organic solvent, polyethylene glycol, and a buffer solution; Preferably, the organic solvent contains at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile; Preferably, the polyethylene glycol contains at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000; Preferably, the buffer solution contains at least one of a citrate buffer solution, a MES buffer solution, a Tris hydrochloride buffer solution, an acetate buffer solution, a disodium hydrogen phosphate–citric acid buffer solution, and a citric acid–sodium hydroxide–hydrochloric acid buffer solution; Preferably, the pH of the buffer solution is 4.2 - 5.0; Preferably, the concentration of the buffer solution is 0.1 - 0.4 M; Preferably, the final concentration of the buffer solution in the simulated solution of the nucleic acid-containing solution is 62 - 88% (v / v); Preferably, the final concentration of the polyethylene glycol in the simulated solution of the nucleic acid-containing solution is 1 - 4% (m / v); Preferably, the final concentration of the organic solvent in the simulation solution of the nucleic acid-containing solution is 10-30% (v / v); Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides; Preferably, in a1) and / or a2), the pushed-out nucleic acid-containing solution or the recovery solution of the nucleic acid-containing solution is recovered by a pipetting device; Preferably, each of the pipetting devices is independently selected from at least one of a pipette, a syringe, and an infusion pump; Preferably, in step a1), the nucleic acid-containing solution contains: DNB; Preferably, the time for rolling circle amplification of the DNB is 20-120 min.

6. The system according to claim 5, wherein: The system includes: A pushing module for pushing the nucleic acid-containing solution, the simulation solution of the nucleic acid-containing solution, the recovery solution of the nucleic acid-containing solution, and / or gas; and A recovery module for recovering the pushed-out nucleic acid-containing solution and / or the recovery solution of the nucleic acid-containing solution; Preferably, the pushing module includes a pipetting device; Preferably, the pushing module further includes a storage container; Preferably, the storage container is used for storing the nucleic acid-containing solution, the simulation solution of the nucleic acid-containing solution, and / or gas; Preferably, the recovery module includes a pipetting device; Preferably, the recovery module further includes a recovery container; Preferably, the recovery container is used for storing the recovery solution of the nucleic acid-containing solution; Preferably, the system is an automated system; Preferably, the devices in the pushing module and / or the recovery module are automated devices.

7. A sequencing method, comprising the following steps: a1) Pushing out the nucleic acid-containing solution in the sequencing groove of the sequencing slide to obtain a recovery solution of the nucleic acid-containing solution; a2) Pushing the recovery solution of the nucleic acid-containing solution obtained in the previous step into the empty sequencing groove of the sequencing slide, incubating, and then pushing out the recovery solution of the nucleic acid-containing solution; 3) Sequencing the nucleic acid in the nucleic acid-containing solution in the sequencing groove of the sequencing slide in a1) and / or a2).

8. The sequencing method according to claim 7, wherein: Step a2) is repeated 0-3 times; Preferably, in step a1), the nucleic acid-containing solution contains any one of DNB, circular DNA, fragmented DNA, a sequencing complex containing a DNB template, a polymerase, and a sequencing primer; Preferably, in step a1), the nucleic acid-containing solution further contains: a loading buffer; Preferably, the number of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times, or more than 30 times the nucleic acid adsorption capacity of the sequencing groove of the sequencing slide.

9. The sequencing method according to claim 8, wherein: The pushing method in step a1) and / or a2) includes a step of pushing out with a simulation solution of the nucleic acid-containing solution; Preferably, the pushing method in step a1) and / or a2) includes the following steps: pushing a simulation solution of the nucleic acid-containing solution into the liquid inlet of the sequencing groove; Preferably, the simulation solution of the nucleic acid-containing solution is pushed in by a pipetting device; Preferably, before pushing a simulated solution containing a nucleic acid solution into the liquid inlet of the sequencing cell, the following steps are further included: pushing a gas into the liquid inlet of the sequencing cell, where the gas does not affect the performance of the nucleic acid in the nucleic acid-containing solution; Preferably, the gas is pushed in through a pipetting device; Preferably, the simulated solution of the nucleic acid-containing solution contains an organic solvent, polyethylene glycol, and a buffer; Preferably, the organic solvent contains at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile; Preferably, the polyethylene glycol contains at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000; Preferably, the buffer contains at least one of citrate buffer, MES buffer solution, Tris hydrochloride buffer, acetate buffer, disodium hydrogen phosphate–citric acid buffer, and citric acid–sodium hydroxide–hydrochloric acid buffer; Preferably, the pH of the buffer is 4.2 - 5.0; Preferably, the concentration of the buffer is 0.1 - 0.4 M; Preferably, the final concentration of the buffer in the simulated solution of the nucleic acid-containing solution is 62 - 88% (v / v); Preferably, the final concentration of the polyethylene glycol in the simulated solution of the nucleic acid-containing solution is 1 - 4% (m / v); Preferably, the final concentration of the organic solvent in the simulated solution of the nucleic acid-containing solution is 10 - 30% (v / v); Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides; Preferably, in a1) and / or a2), the pushed-out nucleic acid-containing solution or the recovered solution of the nucleic acid-containing solution is recovered through a pipetting device; Preferably, the pipetting devices are each independently selected from at least one of a pipette, a syringe, and an injection pump; Preferably, in step a1), the nucleic acid-containing solution contains: DNB; Preferably, the time for rolling circle amplification of the DNB is 20 - 120 min.

10. A sequencing system, the system is used to perform a sequencing method, and the sequencing method includes the following steps: a1) Pushing out the nucleic acid-containing solution in the sequencing cell of the sequencing slide to obtain a recovered solution of the nucleic acid-containing solution; a2) Pushing the recovered solution of the nucleic acid-containing solution obtained in the previous step into the empty sequencing cell of the sequencing slide, incubating, and then pushing out the recovered solution of the nucleic acid-containing solution; 3) Sequencing the nucleic acid in the nucleic acid-containing solution in the sequencing cell of the sequencing slide in a1) and / or a2).

11. The sequencing system according to claim 10, wherein: Step a2) is repeated 0 - 3 times; Preferably, in step a1), the nucleic acid-containing solution contains any one of DNB, circular DNA, fragmented DNA, a sequencing complex containing a DNB template, a polymerase, and a sequencing primer; Preferably, in step a1), the nucleic acid-containing solution further contains: a loading buffer; Preferably, the number of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times, or more than 30 times the nucleic acid adsorption capacity of the sequencing cell of the sequencing slide; Preferably, the pushing method described in step a1) and / or a2) includes a step of pushing out with a simulation solution containing a nucleic acid solution; Preferably, the pushing method described in step a1) and / or a2) includes the following steps: pushing a simulation solution containing a nucleic acid solution into the liquid inlet of the sequencing tank; Preferably, the simulation solution containing a nucleic acid solution is pushed in through a pipetting device; Preferably, before pushing the simulation solution containing a nucleic acid solution into the liquid inlet of the sequencing tank, the following steps are further included: pushing a gas into the liquid inlet of the sequencing tank, and the gas does not affect the performance of the nucleic acid in the nucleic acid solution; Preferably, the gas is pushed in through a pipetting device; Preferably, the simulation solution containing a nucleic acid solution contains an organic solvent, polyethylene glycol, and a buffer solution; Preferably, the organic solvent includes at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile; Preferably, the polyethylene glycol includes at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000; Preferably, the buffer solution includes at least one of citrate buffer solution, MES buffer solution, Tris hydrochloride buffer solution, acetate buffer solution, disodium hydrogen phosphate–citric acid buffer solution, and citric acid–sodium hydroxide–hydrochloric acid buffer solution; Preferably, the pH of the buffer solution is 4.2 - 5.0; Preferably, the concentration of the buffer solution is 0.1 - 0.4M; Preferably, the final concentration of the buffer solution in the simulation solution containing a nucleic acid solution is 62 - 88% (v / v); Preferably, the final concentration of the polyethylene glycol in the simulation solution containing a nucleic acid solution is 1 - 4% (m / v); Preferably, the final concentration of the organic solvent in the simulation solution containing a nucleic acid solution is 10 - 30% (v / v); Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides; Preferably, in a1) and / or a2), the pushed-out nucleic acid solution or the recovery solution of the nucleic acid solution is recovered through a pipetting device; Preferably, the pipetting device includes at least one of a pipette, a syringe, and an infusion pump; Preferably, the nucleic acid solution described in step a1) contains: DNB; Preferably, the time for rolling circle amplification of the DNB is 20 - 120 min.

12. The sequencing system according to claim 11, wherein: A pushing module, which is used for pushing in the nucleic acid solution, the simulation solution of the nucleic acid solution, the recovery solution of the nucleic acid solution, and / or the gas; And A recovery module, which is used for recovering the pushed-out nucleic acid solution and / or the recovery solution of the nucleic acid solution; And A sequencing module, which is used for sequencing the nucleic acid in the nucleic acid solution in the sequencing tank of the sequencing carrier; Preferably, the pushing module includes a pipetting device; Preferably, the pushing module further includes a storage container; Preferably, the storage container is used for storing the nucleic acid solution, the simulation solution of the nucleic acid solution, and / or the gas; Preferably, the recovery module includes a pipetting device; Preferably, the recovery module further includes a recovery container; Preferably, the recovery container is used for storing the recovery liquid of the nucleic acid-containing solution; Preferably, the sequencing module includes a sequencing device; Preferably, the system is an automated system; Preferably, the devices in the pushing module, the recovery module, and / or the sequencing module are automated devices.

13. A method for screening nucleic acids in a nucleic acid-containing solution having specific physical characteristics, comprising the following steps: a1) Pushing out the nucleic acid-containing solution in the sequencing groove of the sequencing slide to obtain a recovery liquid of the nucleic acid-containing solution, wherein the sequencing groove of the sequencing slide can adsorb nucleic acids in the nucleic acid-containing solution having the specific physical characteristics.

14. The method according to claim 13, wherein: The method further comprises the following steps: a2) Pushing the recovery liquid of the nucleic acid-containing solution obtained in the previous step into the empty sequencing groove of the sequencing slide, incubating, and then pushing out the recovery liquid of the nucleic acid-containing solution; Preferably, step a2) is repeated 0-3 times; Preferably, the nucleic acid-containing solution in step a1) comprises any one of DNB, circular DNA, fragmented DNA, a sequencing complex containing a DNB template, a polymerase, and a sequencing primer; Preferably, the nucleic acid-containing solution in step a1) further comprises: a loading buffer; Preferably, the number of nucleic acids in the nucleic acid-containing solution in step a1) is 6 times, 10 times, 15 times, 20 times, or more than 30 times the nucleic acid adsorption capacity of the sequencing groove of the sequencing slide; Preferably, the pushing method in step a1) and / or a2) comprises the step of pushing out with a simulated liquid of the nucleic acid-containing solution; Preferably, the pushing method in step a1) and / or a2) comprises the following steps: Pushing a simulated liquid of the nucleic acid-containing solution into the liquid inlet of the sequencing groove; Preferably, the simulated liquid of the nucleic acid-containing solution is pushed in by a pipetting device; Preferably, before pushing the simulated liquid of the nucleic acid-containing solution into the liquid inlet of the sequencing groove, the following steps are further included: Pushing a gas into the liquid inlet of the sequencing groove, and the gas does not affect the performance of the nucleic acids in the nucleic acid-containing solution; Preferably, the gas is pushed in by a pipetting device; Preferably, the simulated liquid of the nucleic acid-containing solution comprises an organic solvent, polyethylene glycol, and a buffer solution; Preferably, the organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile; Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000; Preferably, the buffer solution comprises at least one of a citrate buffer solution, a MES buffer solution, a Tris hydrochloride buffer solution, an acetate buffer solution, a disodium hydrogen phosphate–citric acid buffer solution, and a citric acid–sodium hydroxide–hydrochloric acid buffer solution; Preferably, the pH of the buffer solution is 4.2-5.0; Preferably, the concentration of the buffer solution is 0.1-0.4 M; Preferably, the final concentration of the buffer solution in the simulated liquid of the nucleic acid-containing solution is 62-88% (v / v); Preferably, the final concentration of the polyethylene glycol in the simulated liquid of the nucleic acid-containing solution is 1-4% (m / v); Preferably, the final concentration of the organic solvent in the simulated solution of the nucleic acid-containing solution is 10-30% (v / v); Preferably, the sequencing slides in a1) and a2) can be the same sequencing slide or different sequencing slides; Preferably, in a1) and / or a2), the pushed-out nucleic acid-containing solution or the recovered solution of the nucleic acid-containing solution is recovered by a pipetting device; Preferably, each of the pipetting devices is independently selected from at least one of a pipette, a syringe, and an injection pump; Preferably, the nucleic acid-containing solution in step a1) contains: DNB; Preferably, the time for rolling circle amplification of the DNB is 20-120 min; Preferably, the empty sequencing slots of the sequencing slide in a2) can adsorb the nucleic acids in the nucleic acid-containing solution having the specific physical characteristics. Preferably, the specific physical characteristics in a1) and a2) can be the same or different; Preferably, the specific physical characteristics in the repeated 2) can be the same or different; Preferably, the physical characteristics include at least one of size, molecular mass, and charge.

15. A simulated solution of a nucleic acid-containing solution, comprising an organic solvent, polyethylene glycol, and a buffer.

16. The simulated solution according to claim 15, wherein: The organic solvent comprises at least one of methanol, ethanol, isopropanol, acetone, and acetonitrile; Preferably, the polyethylene glycol comprises at least one of PEG1000, PEG1500, PEG2000, PE3000, PEG4000, PEG6000, and PEG8000; Preferably, the buffer comprises at least one of a citrate buffer, a MES buffer solution, a Tris hydrochloride buffer, an acetate buffer, a disodium hydrogen phosphate-citric acid buffer, and a citric acid-sodium hydroxide-hydrochloric acid buffer; Preferably, the pH of the buffer is 4.2-5.0; Preferably, the concentration of the buffer is 0.1-0.4 M; Preferably, the final concentration of the buffer in the simulated solution of the nucleic acid-containing solution is 62-88% (v / v); Preferably, the final concentration of the polyethylene glycol in the simulated solution of the nucleic acid-containing solution is 1-4% (m / v); Preferably, the final concentration of the organic solvent in the simulated solution of the nucleic acid-containing solution is 10-30% (v / v).

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